The Role of Protein C in the Regulation of Blood Coagulation
Protein C, a vitamin K dependent plasma protein, is of major physiological importance in the control of hemostasis. Protein C is synthesized as an inactive molecule, herein called nascent protein C. Nascent protein C undergoes complex processing, giving rise to a number of different inactive molecules as is more fully described below. Inactive, secreted forms of protein C are referred to herein as zymogen protein C. Activation of protein C occurs in the blood by a reaction involving a thrombomodulin-thrombin complex. Activated protein C, together with its cofactor protein S, is an anticoagulant of important physiological significance. Activated protein C can prevent intravascular thrombosis and control the extension of existing clots. The mechanism of action of the activated form of protein C and the mechanism of activation of the inactive zymogen into the active protease have been clarified in recent years (for review, see J. E. Gardiner and J. H. Griffin, Progress in Hematology, Vol. XIII, pp. 265-278, ed. Elmer B. Brown, Grune and Stratton, lnc., 1983) and Esmon, N. L. 1989, Prog. Hemost. Thromb. 9:29-55).
The activation of protein C involves thrombin, the final serine protease in the coagulation cascade, and an endothelial cell membrane-associated glycoprotein called thrombomodulin. Thrombomodulin forms a tight, stoichiometric complex with thrombin. Thrombomodulin, when complexed with thrombin, dramatically changes the functional properties of thrombin. Thrombin normally clots fibrinogen, activates platelets, and converts clotting cofactors V and VIII to their activated forms, Va and VIIIa. Finally, thrombin activates protein C, but only very slowly and inefficiently, and the activation is further inhibited by physiological Ca.sup.2+. In contrast, thrombin complexed with thrombomodulin does not clot fibrinogen, activate platelets, or convert clotting factors V and VIII to their activated counterparts Va and VIIIa, but does become a very efficient activator of protein C zymogen in the presence of physiological Ca.sup.2+. The rate constant of protein C zymogen activation by thrombomodulin-thrombin is over 1,000 fold higher than the rate constant for thrombin alone.
To understand how activated protein C down-regulates blood coagulation, the following brief description of the coagulation enzyme system is provided. The coagulation system is best looked at as a chain reaction involving the sequential activation of zymogens into active serine proteases. This chain reaction eventually produces the enzyme thrombin, which through limited proteolysis converts plasma fibrinogen into the insoluble gel fibrin. Two key events in the coagulation cascade are the conversion of clotting factor X to Xa by clotting factor IXa and the conversion of prothrombin into thrombin by clotting factor Xa. Both of these reactions occur on cell surfaces, most notably the platelet surface, and both reactions require cofactors. The major cofactors, factors V and VIII, in the system circulate as relatively inactive precursors, but when the first few molecules of thrombin are formed, thrombin loops back and activates the cofactors through limited proteolysis. The activated cofactors, Va and VIIIa, accelerate both the conversion of prothrombin into thrombin and also the conversion of factor X to factor Xa by approximately five orders of magnitude. Activated protein C preferentially acts on, to proteolytically degrade, hydrolyze, and irreversibly destroy clotting cofactors Va and VIIIa, the activated forms of the inactive clotting factors V and VIII. Clotting factors V and VIII, in contrast, are very poor substrates for activated protein C in vivo.
An important cofactor for activated protein C is protein S, another vitamin K-dependent plasma protein. Protein S substantially increases activated protein C-mediated hydrolysis of factors Va and VIIIa 25 fold.
Protein C as a Therapeutic Agent
Protein C is recognized as a valuable therapeutic agent (see, for example, Bang et al., U.S. Pat. No. 4,775,624, issued Oct. 4, 1988, the teaching of which is incorporated herein by reference). Activated protein C is a novel antithrombotic agent with a wider therapeutic index than available anticoagulants, such as heparin and the oral hydroxycoumarin type anticoagulants. Neither zymogen protein C nor activated protein C is effective until thrombin is generated, because thrombin is needed to convert clotting factors V to Va and VIII to VIIIa; the activated forms of these two cofactors are the preferred substrate for activated protein C. Thrombin is also required to activate zymogen protein C, for without the thrombomodulin-thrombin complex, the protein C zymogen is not efficiently converted into its active counterpart.
Activated protein C is an on-demand anti-coagulant, because activated protein C works by inactivating cofactors Va and VIIIa. Because thrombin is required to convert factors V and VIII to their activated counterparts Va and VIIIa, protein C only acts as an anticoagulant after thrombin is generated. Conventional anticoagulants, in contrast to activated protein C, maintain a constant anticoagulant state throughout the circulation for as long as they are given to the patient, thereby substantially increasing the risk of bleeding complications over that for protein C or activated protein C. Activated protein C is therefore an on-demand anticoagulant of wide clinical utility for use as an alternative to heparin and the hydroxycoumarins.
In some disease states, such as hereditary protein C deficiency, protein C zymogen is of great therapeutic importance. In congenital homozygous protein C deficiency, affected individuals die in early childhood from purpura fulminans, an often lethal form of disseminated intravascular coagulation. In heterozygous protein C deficiency, affected individuals suffer severe, recurrent thromboembolic episodes. It is well established clinically that plasma protein concentrates designed to treat hemophilia B or factor IX deficiency, which contain protein C as an impurity, are effective in the prevention and treatment of intravascular clotting in heterozygous protein C deficiency. Protein C levels have also been noted to be abnormally low in thrombotic states such as disseminated intravascular coagulation and in disease states predisposing to thrombosis, such as major trauma, major surgery, and cancer.
The Synthesis and Activation of Human Protein C
To facilitate an understanding of the activation of protein C and the invention, the coding sequence, and corresponding amino acid residue sequence, for nascent human protein C is depicted below. This amino acid residue sequence, and relevant portions thereof, also characterizes "native human protein C" for purposes of the present invention.
wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytidyl, T is thymidyl, ALA is Alanine, ARG is Arginine, ASN is Asparagine, ASP is Aspartic acid, --COOH is the carboxy terminus, CYS is Cysteine, GLN is Glutamine, GLU is Glutamic Acid, GLY is Glycine, HIS is Histidine, H.sub.2 N- is the amino terminus, ILE is Isoleucine, LEU is Leucine, LYS is Lysine, MET is Methionine, PHE is Phenylalanine, PRO is Proline, SER is Serine, THR is Threonine, TRP is Tryptophan, TYR is Tyrosine, and VAL is Valine.
The DNA sequence depicted above was derived from cDNA clones prepared from human liver mRNA that encodes human protein C. Those skilled in the art recognize that the degenerate nature of the genetic code enables one to construct many different DNA sequences that encode the same amino acid residue sequence. The cDNA sequence for nascent human protein C depicted above is thus only one of many possible nascent human protein C-encoding sequences. In constructing the cDNA clones, a 5' poly G sequence, a 3' poly C sequence, and both 5' and 3' PstI restriction enzyme recognition sequences were constructed at the ends of the protein C-encoding cDNA. Two of these cDNA clones were manipulated to construct a DNA molecule comprising both the coding sequence of nascent human protein C and also portions of the DNA encoding the untranslated mRNA at the 5' and 3' ends of the coding region. This DNA molecule was inserted into the PstI site of plasmid pBR322 to construct plasmid pHC7. Plasmid pHC7 thus comprises the coding sequence above and, again depicting only one strand of the molecule, also contains these additional sequences: ##STR1## at the 5' and 3' ends, respectively, of the coding strand of the nascent human protein C coding sequence. Due to the complementary nature of DNA base-pairing, the sequence of one strand of a double-stranded DNA molecule is sufficient to determine the sequence of the opposing strand. Plasmid pHC7 can be conventionally isolated from E. coli K12 RR1/pHC7, a strain deposited with and made part of the permanent stock culture collection of the Northern Regional Research Laboratory (NRRL), Peoria, Ill. A culture of E. coli K12 RR1/pHC7 can be obtained from the NRRL under the accession number NRRL B-15926. A restriction site and function map of plasmid pHC7 is presented in FIG. 2 of the accompanying drawings.
Nascent protein C can also be depicted schematically, as shown below. ##STR2## pre-pro--amino acid residues 1-42 of nascent human protein C encode the signal peptide and pro--peptide of human protein C, important for directing secretion and .gamma.-carboxylation of protein C.
LC--amino acid residues 43-197 of nascent protein C, once post--translationally modified, constitute the light chain (LC) of both the two-chain zymogen (formed from one-chain zymogen by removal of the KR dipeptide, as discussed below) and activated forms of protein C. PA1 KR--amino acid residues 198-199 of nascent human protein C; these residues are believed to be removed (on the basis of homology with bovine protein C), probably by a two-step process comprising a first cleavage (either between residues 197-198 or 199-200) followed by carboxypeptidase or aminopeptidase action, to form two-chain protein C. PA1 AP--amino acid residues 200-211 of nascent protein C constitute the activation peptide, which is removed from the zymogen forms of protein C to obtain activated protein C. PA1 AHC--amino acid residues 212-461 of nascent protein C, once post-translationally modified, constitute the activated heavy chain (AHC) of active protein C. PA1 HC--the heavy chain of the two chain form of protein C zymogen, once post-translationally modified, is composed of amino acid residues 200-461, the AP and AHC. PA1 Ad2LP--the major late promoter of adenovirus type 2. PA1 Amino acid residues in proteins or peptides described herein as abbreviated as follows in Table I. PA1 ApR--the ampicillin--resistant phenotype or gene conferring same. PA1 BK--DNA from BK virus. PA1 Enh or enhancer--the enhancer of BK virus. PA1 ep or SV40ep--a DNA segment comprising the SV40 early promoter of the T-antigen gene, the T-antigen binding sites, the SV40 enhancer, and the SV40 origin of replication. PA1 .gamma.-carboxylation--a reaction which adds a carboxyl group to glutamic acids at the .gamma.-carbon. PA1 .gamma.-carboxylated protein--a protein in which some glutamic acids residues have undergone .gamma.-carboxylation. PA1 GBMT transcription unit--a modified transcription control unit comprising the P2 enhancer of BK virus spaced closely to the upstream regulatory element of the adenovirus major late promoter (MLTF), the adenovirus-2 major late promoter, a poly GT element positioned to stimulate said promoter and a DNA sequence containing the spliced tripartite leader sequence of adenovirus. The GBMT transcription unit is found on an approximately 900 base pair HindIII restriction fragment of plasmid pGT-h. PA1 IVS--DNA encoding an intron, also called an intervening sequence. PA1 MMTpro--the promoter of the mouse metallothionein-I gene. PA1 Nascent protein--the polypeptide produced upon translation of a mRNA transcript, prior to any post-translational modifications. However, post-translational modifications such as .gamma.-carboxylation of glutamic acid residues and hydroxylation of aspartic acid residues may begin to occur before a protein is fully translated from an mRNA transcript. PA1 NeoR--a neomycin resistance-conferring gene, which can also be used to confer resistance to the antibiotic G418. PA1 pA--a DNA sequence encoding a polyadenylation signal. PA1 Promoter--a DNA sequence that directs transcription of DNA into RNA. PA1 Protein C activity--any property of human protein C responsible for proteolytic, amidolytic, esterolytic, and biological (anticoagulant or profibrinolytic) activities. Methods for testing for protein anticoagulant activity are well known in the art, i.e., see Grinnell et al., 1987, Biotechnology 5:1189. PA1 Recombinant DNA Cloning Vector--any agent, including, but not limited to; chromosomally integrating agents, autonomously replicating plasmids, and phages, comprising a DNA molecule to which One or more additional DNA segments can be or have been added. PA1 Recombinant DNA Expression Vector--any recombinant DNA cloning vector into which a promoter has been incorporated and positioned to drive expression of a gene product. PA1 Recombinant DNA Vector--any recombinant DNA cloning or expression vector. PA1 Replicon--A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector. PA1 Restriction Fragment--any linear DNA sequence generated by the action of one or more restriction endonuclease enzymes. PA1 Sensitive Host Cell--a host cell that cannot grow in the presence of a given antibiotic or other toxic compound without a DNA segment that confers resistance thereto. PA1 TcR--the tetracycline--resistant phenotype or gene conferring same. PA1 Transformation--the introduction of DNA into a recipient host cell that changes the genotype of the recipient cell. PA1 Transformant--a recipient host cell that has undergone transformation. PA1 Translational Activating Sequence--any DNA sequence, inclusive of that encoding a ribosome binding site and translational start codon, such as 5'-ATG-3', that provides for the translation of a mRNA transcript into a peptide or polypeptide. PA1 Zymogen--an enzymatically inactive precursor of a proteolytic enzyme. Protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chain, of protein C.
Human protein C zymogen is a serine protease precursor synthesized in the liver and present in the blood. For expression of complete biological activity, protein C requires post--translational modifications for which vitamin K is needed. The two-chain, disulfide-linked, protein C zymogen arises from the single-chain zymogen by limited proteolysis. This limited proteolysis is believed to include cleavage and removal of amino acid residues 198 and 199. The activation of the two-chain zymogen into the active serine protease involves the proteolytic cleavage of an ARG-LEU peptide bond (residues 211 and 212). This latter cleavage releases a dodecapeptide (residues 200-211), the activation peptide, that constitutes the amino-terminus of the larger (heavy) chain of the two-chain zymogen molecule. Protein C is significantly glycosylated; the mature enzyme from plasma reportedly contains 15 to 23% carbohydrate. Protein C also contains a number of unusual amino acids, including .gamma.-carboxyglutamic acid and .beta.-hydroxyaspartic acid (erythro-L-.beta.-hydroxy aspartate). .gamma.-carboxyglutamic acid (gla) is produced by .gamma.-glutamyl carboxylation from glutamic acid residues with the aid of a hepatic microsomal carboxylase which requires vitamin K as a cofactor.
The activation of human protein C can also be represented schematically and is shown below. Those skilled in the art recognize that the order of the steps shown in the schematic do not necessarily reflect the order of the steps in the in vivo pathway. ##STR3## The present invention provides novel compounds, vectors, transformants, and methods for the recombinant expression of novel protein C zymogens.